ON-treatment Single-cell Analysis for the Identification of Early Tumor Response Biomarkers on Prospective Collected Serial Tumor Biopsies in Triple Negative Breast Cancer Patient During Standard Neoadjuvant Chemo-immunotherapy

This exploratory translational study investigates early breast cancer (EBC), focusing on triple-negative (TNBC) and high-risk luminal (ER+/HER2-) subtypes. By integrating single-cell RNA sequencing, using the 10x Genomics platform (or similar technologies if it will be not available at the time of experiment), and spatial imaging analyses from serial tumor biopsies, the project aims to characterize tumor-immune interactions and identify predictive biomarkers of response or resistance to neoadjuvant chemo-immunotherapy.

We hypothesize that early molecular and spatial features, when detected at the single-cell level, can predict treatment sensitivity or resistance. This would enable adaptive therapeutic strategies that minimize overtreatment while maximizing efficacy in EBC. Our goal is to reveal, for the first time, the cellular heterogeneity and spatial interactions that drive therapy resistance and disease progression.

The analysis will be performed on tumor biopsies collected before the start of treatment, after C1D1 and on surgical material. The only tissue sample collected specifically for the study is the biopsy after C1D1.

Inclusion criteria

• Female patients aged ≥18 years.
• ECOG performance status 0-1.
• Histologically confirmed early breast cancer with one of the following molecular profiles:

• TNBC: ER and PR negative (IHC <10%) and HER2 negative (IHC 0-1+ or FISH non-amplified).
• High-risk luminal (ER+/HER2-): ER positive (IHC ≥10%) HER2 negative (IHC 0-1+ or FISH non-amplified), with high-risk features (e.g., Grade 3, PR-negative, high proliferation, high TILS).
• Clinical indication for neoadjuvant treatment according to standard practice:

• TNBC: cT1c and/or cN positive, or cT2 (>2 cm) and/or cN positive (stage II,III).
• High-risk luminal: features as defined above (Grade 3, PR-negative, high proliferation, ER low).
• Ability to understand and sign written informed consent for participation in the study, approved by the local Ethics Committee.

Exclusion criteria

• HER2-positive tumors.
• Multifocal tumors - exclusion if a single index lesion cannot be identified and sampled; otherwise allowed if a representative lesion can be biopsied.
• Known metastatic disease. 4 Clinical contraindications to the planned neoadjuvant therapy.

• Decision for upfront surgery as determined by the multidisciplinary team. 6. Inability to provide informed consent. 7. Pregnancy or breastfeeding. 8. Prior systemic therapy (chemotherapy, immunotherapy, or endocrine therapy) for the current breast cancer before baseline biopsy 9. On-treatment biopsy clinically not feasible or controindicated